SUMMARY Visceral pain is notoriously difficult to treat, often persisting long after the precipitating injury/disease is no longer evident. In this application we will explore a novel, multicellular peripheral circuit that we hypothesize explains many of the intractable features of chronic, visceral pain. We now know that epithelial-neuronal communication is widespread, with numerous epithelial cell types releasing neuroactive substances (e.g., ATP, ACh, 5HT, glutamate). This is particularly apparent in the colon where we have found that channelrhodopsin (ChR2) -induced activation of colon epithelial cells produces high frequency bursting of colon extrinsic primary afferent neurons (ExPAN?s), phenocopying physiologic stimuli and inducing robust behavioral responses (visceromotor responses (VMR), a validated assay of hypersensitivity). Building on these findings, new surprising data indicate colon epithelium also receives functional input from sympathetic neurons; activation of sympathetic projections to the colon induces large, phase-locked calcium signals in the epithelium. Closing the loop, we found that activation of ExPAN?s via colorectal distension (CRD) induces calcium signals in the post-ganglionic sympathetic neurons projecting to the colon, and that ChR2- induced activation of ExPAN?s induces cFos expression in these same neurons. That this multicellular circuit plays a role in visceral pain is supported further by preliminary data that shows that inflammation (acute and/or chronic) is correlated with increased signaling in all portions of this circuit. Thus, the goal of the proposed experiments is to test the hypothesis that persistent visceral hypersensitivity is due, at least in part, to amplification in an epithelial-ExPAN-sympathetic circuit such that it is possible to treat pain by breaking any limb of this feed-forward circuit (Fig.1). This hypothesis will be tested in 3 aims: Aim 1: Determine if persistent hypersensitivity induced in a model of IBD (DSS (dextran sulfate sodium)) is due to increased epithelial signaling and/or ExPAN excitability, Aim 2: Determine if DSS-induced inflammation increases the ability of ExPANs to activate sympathetic neurons in prevertebral sympathetic ganglion (PrSG) directly (via synapses in PrSG) or indirectly (via a spinal cord circuit) and, Aim 3 Determine the ability of sympathetic neurons to drive activity in epithelial cells in nave mice and in the DSS model of IBD.